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NEUROENDOCRINOLOGY |
School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom
Address all correspondence and requests for reprints to: Dr. Simon Luckman, 1.124 Stopford Building, School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom. E-mail: simon.luckman{at}man.ac.uk
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Abstract |
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Introduction |
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Recently, we have proposed an alternative role for PrRP in appetite regulation (15). Intracerebroventricular (icv) injection of PrRP causes a reduction in food intake in both free-feeding and fasted rats. The concomitant decrease in body weight gain is not due entirely to the reduction in food intake because pair feeding experiments indicate additional effects on energy expenditure. PrRP mRNA, in each of the three locations where it is expressed, the dorsomedial hypothalamic nucleus (DMH), the brainstem nucleus of the tractus solitarius (NTS), and the ventrolateral medulla (VLM), is reduced in two rat models of negative energy balance, fasting, and lactation, providing strong evidence for a physiological role for the peptide in the regulation of food intake.
Here we demonstrate the activation of specific brain regions by centrally administered PrRP using c-fos functional mapping. We compared the action of PrRP to the satiety factor cholecystokinin (CCK) and a nonhomeostatic inhibitor of food intake, lithium chloride (LiCl). Both CCK and LiCl act on central circuits via catecholaminergic neurons of the brainstem (16, 17), and it is believed that the two agents may use the same pathways leading to reduced food intake and delayed gastric emptying (15, 18). Indeed both LiCl and high doses of CCK can cause conditioned taste aversion (CTA) (19, 20), suggesting that the expression of satiety and visceral illness may represent two extreme manifestations of the same sensation. We investigated whether PrRP reduced food intake by inducing visceral illness and determined its effect on the normal behavioral satiety sequence (BSS). The normal sequence of events observed in rodents housed singly, which are first denied access to food and then allowed to eat, consists of the termination of eating, followed by exploratory behaviors, grooming, and finally rest or sleep (21, 22). Lastly, we provide evidence that PrRP neurons in the brainstem may mediate selectively peripheral satiety signaling.
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Materials and Methods |
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PrRP (Bachem, Saffron Walden, UK, or Peptide, Inc., Japan) was dissolved in 0.1% BSA/saline or sterile water. CCK (CCK 26–33, sulfated, Sigma-Aldrich Corp. Ltd., Poole, UK) was dissolved in isotonic saline (0.15 M NaCl) and LiCl (Sigma-Aldrich Corp.) was dissolved in water (to make an isotonic, 0.15 M, solution).
Exp 1: food intake and PrRP
PrRP (4 nmol in 2 µl, n = 5) or vehicle (2 µl water, n = 5) was injected icv into 24 h food-deprived rats, 2 h after the beginning of the light phase. A preweighed amount of food was presented subsequently to the animals, and food consumption was measured at 1, 2, 4, and 8 h after injection. Body weight was measured before and 8 h after injection.
Exp 2: CTA test
PrRP, CCK, and LiCl were tested using a one-bottle CTA test in three separate experiments. Animals that had been water deprived overnight, were provided with a novel 0.05 M sucrose solution the following morning (d 1) for 20 min. The amount of sucrose solution consumed was measured, then replaced with water, and the rats received either an icv injection of PrRP (4 nmol in 2 µl), or an ip injection of CCK (50 µg/kg) or LiCl (0.15 M, 20 ml/kg). Control animals received water (icv, 2 µl) or 0.15 M NaCl (20 ml/kg, ip), respectively (n = 5–7 for each of treatment groups). On d 2, the rats were again water deprived overnight, and the following morning (d 3) were exposed again to 0.05 M sucrose solution for 20 min, and the amount consumed was recorded. Data are presented as amount (in ml) of sucrose solution ingested in 20 min on d 3 (i.e. on second exposure).
Exp 3: behavioral satiety sequence
Animals were habituated to transparent cages 1.5 h/d for 2 d before the experiment. PrRP (4 nmol, icv, n = 14), or in a separate experiment CCK (50 µg/kg, ip, n = 12) or LiCl (0.15 M, 20 ml/kg, ip, n = 6) was administered into animals that had been fasted overnight. Control rats (both groups n = 13) received equivalent volumes of their respective vehicles. After injections, a preweighed amount of food was placed in the cages, and the animals were left undisturbed for 90 min. Behavior was recorded every 30 sec for 90 min, at which point food intake was measured. Behavior was classified into: feeding (animal at hopper trying to obtain food, chewing, gnawing or holding food in paws), drinking (animal licking spout of water bottle), grooming (animal scratching, licking or biting any part of its anatomy), resting (animal curled up, resting head with eyes closed), or other (any other behavior or activity, including locomotion, sniffing, rearing, immobility when aware, or signs of sickness behavior). Data were collated into 5-min period bins for display. Three variables were analyzed: food intake, latency to rest (i.e. time at which animals first displayed resting), and the transition from eating to resting (i.e. the time at which the frequency of eating within the group matches the frequency of resting).
Exp 4: c-Fos immunohistochemistry
In separate experiments, rats received either an icv injection of PrRP (4 nmol) or an ip injection of CCK (50 µg/kg) or LiCl (0.15 M, 20 ml/kg). Control groups received equal volumes of vehicle by the appropriate route. Ninety minutes after treatments, rats were anesthetized terminally with an overdose of sodium pentobarbitone (ip; Rhône Mérieux, Harlow, UK) and perfused transcardially with 4% paraformaldehyde. Equivalent 30-µm sections, 90 µm apart, were cut on a sledge microtome. Sections were collected according to the atlas of Paxinos and Watson (23) through the forebrain at the level of the hypothalamus (bregma -0.3 mm to -4.5 mm) and the caudal brainstem (bregma -12.8 mm to -14.6 mm). Free-floating sections were incubated in a rabbit polyclonal anti-c-Fos antibody (1:1000; sc-52, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and then a peroxidase-labeled goat antirabbit IgG antibody (1: 500; Vector Laboratories, Inc. Burlingame, CA). Nuclear c-Fos was visualized using a nickel-intensified diaminobenzidine reaction to produce a black precipitate. For the CCK and LiCl experiments, the sections were then incubated sequentially in a rabbit polyclonal antibody raised against the human PrRP sequence (1:4000; Phoenix Pharmaceuticals, Inc., Belmont, CA), biotinylated antirabbit IgG (1:200; Vector Laboratories, Inc., Burlingame, CA) and then a streptavidin-biotin-peroxidase complex (1:200; Amersham Pharmacia Biotech, Little Chalfont, UK). Cytoplasmic PrRP staining was visualized by a normal diaminobenzidine reaction to yield a brown precipitate. In the PrRP experiment, the number of neurons expressing c-Fos was counted bilaterally in nuclei defined by the atlas of Paxinos and Watson (23) and with reference to counterstained adjacent sections. In the CCK/LiCl experiment, c-Fos was only counted on sections where PrRP neurons are located (that is level with and caudal to the area postrema in the brainstem, and in the caudal DMH). The mean number of neurons per section was determined for each animal, and then the group mean was determined (n = 7 per group for PrRP experiment, n = 5 per group for CCK experiment and n = 6 per group for the LiCl experiment).
Statistical analyses
All data are presented as mean ± SEM. Data from two groups were analyzed using an unpaired t test or a Mann-Whitney U test where appropriate, and for three groups parametric analysis of variation or the nonparametric Kruskal-Wallis test. Statistical significance was taken where P < 0.05.
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Results |
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), whereas ingestion of water was unaffected (data not shown). The decrease in food intake was accompanied by a reduction in body weight gain (control 4 ± 1 g vs. PrRP -1 ± 2 g, P < 0.05, measured at the 8 h time point) compared with vehicle-treated group.
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); animals conditioned by LiCl drank significantly less sucrose solution on d 3 compared with the group that had received a control saline injection (73% reduction; P < 0.001). CCK caused a slight reduction in sucrose drinking on d 3 (35%; P < 0.05). PrRP, at the dose shown to reduce food intake, did not support CTA (comparison of sucrose drunk on d 3; Fig. 2, P > 0.05).
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, A and D). Though the sequence was more delayed in icv-treated compared with ip-treated animals, it is not possible to directly compare different treatment routes. LiCl and CCK, ip, reduced the amount of food eaten over the test period (control 3.9 ± 0.4 g, LiCl 0.1 ± 0.1 g, CCK 2.3 ± 0.2 g; P < 0.001 or P < 0.05 for LiCl or CCK, respectively, compared with control). The BSS was completely disrupted in LiCl-treated rats, which showed extended periods of immobility without resting (Fig. 3B). These rats did not sleep within the test period. Figure 3C reveals that the normal BSS is maintained in CCK-treated animals. Furthermore, the point of transition from eating to resting took place in time bin 7 in the CCK group, compared with bin 11 in the equivalent control group (Fig. 4, A and C). However, there was no statistically significant difference in the average latency to rest (control 58 ± 6 min, vs. CCK 51 ± 9 min; P > 0.05). PrRP reduced food intake over the test period (control 6.8 ± 0.4 g vs. PrRP 4.0 ± 0.3 g; P < 0.001) but did not disrupt the normal BSS (Fig. 3E) when compared with control (Fig. 3D). As with CCK, there was an apparent shift of the sequence to the left. While the transition from eating to resting did not occur within the 90–0 min test period for the control group, the transition occurred in time bin 11 in the PrRP-treated group (Fig. 4, D and E). There was a significant reduction in the latency to rest in animals injected with PrRP (control 83 ± 4 min vs. PrRP 51 ± 6 min; P < 0.01).
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, Table 1). The induction of c-Fos in the hypothalamic paraventricular nucleus was mostly in its parvocellular regions and that in the area postrema was mainly in the inner peripheral margin of the structure. The average number of c-Fos-positive profiles in the supraoptic nucleus and the DMH of the hypothalamus and in the bed nucleus of the stria terminalis was higher in each case, but these changes did not quite reach statistical significance. No induction of c-Fos was noted in the thalamic reticular nucleus or in the hypothalamic periventricular and arcuate nuclei.
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, B and E). This was a strong stimulus for brainstem PrRP neurons, the majority of which expressed c-Fos (for the rostrocaudal extent of the population: NTS, 7 ± 3% after vehicle to 84 ± 4% after CCK; VLM, 9 ± 5% to 65 ± 4%; both P < 0.001). However, there was some evidence for regional differences in the response, as less than half of the PrRP neurons caudal to the area postrema expressed c-Fos following CCK, whereas most of those adjacent to the area postrema did (results not shown). There was no significant increase in the number of cells expressing c-Fos in the DMH (vehicle 24 ± 5 vs. CCK 48 ± 13; P > 0.05). Though some PrRP neurones did express c-Fos, this did not change significantly following treatment (20 ± 3% in the vehicle group and 15 ± 1% in the CCK group). LiCl increased the expression of c-Fos in each of the three brain areas on sections where PrRP neurons are located (NTS, 7 ± 2 to 75 ± 5; VLM 3 ± 1 to 12 ± 1; DMH 20 ± 5 to 83 ± 8, all P < 0.01). However, in none of these areas was there a change in the percentage of PrRP neurons expressing c-Fos (NTS, 8 ± 1% in the vehicle group and 7 ± 1% in the LiCl group; VLM, 4 ± 1% and 7 ± 1%; DMH, 3 ± 1% and 2 ± 1%, all P > 0.05, Fig. 6).
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Discussion |
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LiCl caused a reduction in food intake due to visceral illness and nausea, as demonstrated here by the disruption of the normal BSS and the production of CTA. By comparison, and as previously described (21), CCK also reduced food intake but supported a normal BSS. The dose of CCK used in the present experiments was chosen as it reduces food intake and causes a robust induction of c-Fos protein in the brain (16, 25). However, this dose may have caused some nausea in our animals as a slight CTA was produced. This is consistent with previous published work that has demonstrated that high doses of CCK can produce nausea (for review see Ref. 20). PrRP reduced food intake, but did not cause CTA or disrupt the BSS, suggesting that it may function as a homeostatic regulator of food intake. There was a shift in the BSS resulting in a reduced latency to rest, as might be expected by a true satiety factor (22).
Both homeostatic and nonhomeostatic regulators of food intake can stimulate hypothalamic neurons which produce CRH and oxytocin (18, 24). PrRP caused the activation of cells in the parvocellular portion of the hypothalamic paraventricular nucleus, which presumably includes neurons containing CRH and oxytocin. There was also a trend toward an increase of c-Fos in the supraoptic nucleus, where magnocellular oxytocin neurons reside. As with systemic injection of CCK (25), doses of PrRP higher than those used in the present study produce the secretion of both oxytocin and ACTH (12, 14). By contrast, two other regions of the forebrain activated by PrRP, the paraventricular nucleus of the thalamus and the dorsal lateral hypothalamic area, have not been reported to be stimulated by CCK. Interestingly, however, both of these areas have been implicated in appetite regulation (28, 29).
The induction of c-Fos observed here overlaps partially with regions that contain PrRP-immunoreactive fibers or PrRP receptor mRNA. Two areas of the forebrain that display high levels of expression of the PrRP receptor mRNA and PrRP binding, the thalamic reticular nucleus and the hypothalamic periventricular nucleus (5, 9, 15, 30) did not express c-Fos after PrRP treatment. This may be because PrRP inhibits neuronal activity in these areas. As both of these regions contain populations of inhibitory neurons [GABA or somatostatin (31, 32)], some of the stimulatory effects of PrRP in other regions may be due to indirect disinhibition.
It is established that signals from the gastrointestinal tract concerned with both satiety and visceral illness are detected by vagal nerve afferents that project to the brainstem NTS (19, 26). Central processing of these stimuli can be detected as the induction of c-Fos in both the brainstem and forebrain structures (16, 17, 24, 27). We have shown previously that, in the brainstem, approximately half of the neurons that are induced to express c-Fos following CCK are noradrenergic neurons of the A1 and A2 cell groups (16) and a proportion of these project to the hypothalamus (33, 34). Other activated neurons in the brainstem may be interneurons, or those responding to pathways descending from the hypothalamus (35). Conversely, only half of the total number of noradrenergic neurons in the NTS and VLM regions are activated by this stimulus, suggesting that subpopulations of A1 and A2 cells are involved (16). Attempts to further characterize the phenotype of neurons, perhaps activated by CCK but not by LiCl, have not defined specific subpopulations (36, 37). Here we report that CCK activated a large proportion of PrRP neurons in the NTS and VLM, whereas LiCl did not. Because PrRP, in both the NTS and VLM, colocalizes with tyrosine hydroxylase (2, 30, 38, 39, 40), this is the first indication that a subpopulation of brainstem noradrenergic cells may respond selectively to different stimuli. The effect on PrRP neurons contrasts to the situation with glucagon-like peptide 1 (GLP-1)-containing neurons in the brainstem that are stimulated by both CCK and LiCl (37). It has been argued that GLP-1 neurons of the brainstem may mediate the effects of visceral illness rather than satiety (41, 42).
It will be interesting to map afferent projections to each of the forebrain areas activated by PrRP to determine whether they are directly innervated by ascending brainstem neurons, or by PrRP neurons of the DMH that did not respond acutely to CCK injection but which are regulated by energy status (15). It is possible that PrRP, GLP-1, and other peptidergic neurons display complex responses to different forms and intensity of gut signaling. In addition, we cannot rule out that PrRP in the brainstem may have other roles, for example in blood pressure regulation or in mediating other forms of stress (10, 40).
In summary, PrRP causes a reduction in free-feeding and fast-induced feeding without affecting water intake (15). It is unlikely that PrRP reduces food intake due to nonspecific or nonhomeostatic actions because it did not support CTA or disrupt the normal BSS. Furthermore, a reduction in the expression of PrRP mRNA in the brain following fasting or during lactation, two states in which the animal is in negative energy balance (15), is a strong indication that PrRP has a physiological role to play in appetite and body weight control. The activation of brainstem PrRP neurons by CCK, but not by LiCl, provides evidence to suggest that while there may be common pathways between the gut and the brain mediating satiety and visceral illness, there may also be some parallel but selective components. This leads to the possibility that some of the central effects of CCK on satiety are mediated by PrRP.
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Acknowledgments |
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Footnotes |
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Abbreviations: BSS, Behavioral satiety sequence; CCK, cholecystokinin; CTA, conditioned taste aversion; DMH, dorsomedial hypothalamic nucleus; GLP-1, glucagon-like peptide 1; icv, intracerebroventricular(ly); LiCl, lithium chloride; NTS, tractus solitarius; PrRP, PRL-releasing peptide; VLM, ventrolateral medulla.
Received August 16, 2001.
Accepted for publication October 17, 2001.
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